(1) To determine the functional impact of recurrent FBXW7 and SPOP mutations uncovered in endometrial cancers Ubiquitin-mediated proteasomal degradation plays a critical role in maintaining proper levels of cellular proteins, including proteins that promote tumorigenesis. The degradation process is tightly regulated, and is mediated by a large number of substrate-specific E3 ubiquitin ligases including the SKP1-CUL1-FBXW7 and SPOP-CUL3-RBX1 ubiquitin ligases. In previous work, we discovered that FBXW7 and SPOP are highly mutated in serous and clear cell endometrial carcinomas, with most mutations residing in the substrate-binding regions of the encoded proteins (Le Gallo et al., Nature Genetics 2012). We hypothesize that the mutations act in a loss-of-function or dominant-negative manner to improperly regulate the proteasomal degradation of one or more substrates. We are currently testing this hypothesis in ongoing functional studies using biochemical and cell-based approaches. We are focusing our efforts on recurrent mutations in these genes, as well as unique mutations that are predicted (in silico) to impact protein function. Progress in the current reporting period is as follows: (1a) Using shRNA knockdown and Western blotting, we have identified several substrates of FBXW7 that exhibit increased protein levels upon knockdown of FBXW7 in serous endometrial cancer cell lines. We have found that a number of these substrates also reproducibly show increased protein levels upon transient overexpression of mutant FBXW7 constructs compared to wildtype FBXW7 constructs in endometrial cancer cells. These findings suggest that the mutant forms of FBXW7 may be functionally altered and unable to properly regulate the turnover of key protein substrates. We are currently attempting to generate stable cell lines expressing these FBXW7 wildtype and mutant constructs and we are actively looking into the feasibility of using CRISPR/Cas9 technology to stably introduce FBXW7 mutations into endometrial cancer cells, to provide a more defined system for longer-term functional studies. (1b) Our ongoing studies of SPOP have assessed the ability of SPOP mutations to regulate an SPOP-CUL3-RBX1 protein substrate upon transient expression in the HEK293 cell line. Specifically, we have transiently expressed thirteen mutants of interest, as well as a known loss-of-function mutant of SPOP that serves as an internal control, in HEK293 cells. We have reproducibly shown that three of the thirteen SPOP mutants correlate with increased levels of a specific substrate protein in transiently transfected cells. In parallel, we have used co-immunoprecipitation and immunoblotting to determine whether the observed increase in substrate levels might result from impaired SPOP-substrate binding. In ongoing experiments that will extend into the next reporting period, we are assessing the potential cellular effects of the three SPOP mutants in viability assays, clonogenic assays, and wound healing assays. A poster describing the interim findings of the research within this aim was presented at the 2016 annual meeting of the American Association for Cancer Research by Postdoctoral Fellow Dr. Fred Lozy. (2) To identify somatically mutated driver genes for clear cell endometrial carcinomas This aim is a continuation, and near finalization, of work that was initiated and brought close to completion in previous reporting periods. The strategy we used to identify somatically mutated genes in clinically diagnosed clear cell endometrial carcinomas, in previous reporting periods, employed whole exome sequencing of 16 paired tumor-normal DNAs. Sequence variants identified within the tumor exomes were extensively curated by former Postdoctoral Fellow Dr. Matthieu Le Gallo to identify somatic (tumor-specific) variant calls. The somatic variant calls were further evaluated by Sanger sequencing to uncover a set of true (validated) somatic mutations. In previous reporting periods we Sanger sequenced selected genes (genes-of-interest) from a large cohort of clinically diagnosed clear cell endometrial tumors provided via national and international collaborators. In the current reporting period, we reanalyzed a subset of the data, and prepared a manuscript describing the major findings of this study. We are currently conducting pathological review of a subset of cases. We aim to submit this manuscript to a peer-reviewed scientific journal in the near future. (3) To identify somatically mutated genes that drive advanced-stage endometrioid endometrial cancers In the current reporting period we extracted and purified genomic DNAs from 19 advanced stage (stage III or IV) primary endometrioid endometrial tumor tissues as well as matched non-tumor tissues from the same patients. The purified DNAs have been whole exome sequenced by the NIH Intramural Sequencing Center. The Roche SeqCap EZ Exome+UTR Library capture kit was used for exome capture and the captured libraries were sequenced on Illumina HiSeq2500 instruments. The resulting short sequence reads for each exome have been aligned to the human reference sequence to assess a number of parameters including, but not limited to, the percentage of target regions covered, the average read depth for target regions, the total number of within-target sequence variants, and the number of within-target sequence variants that are in build 37 of dbSNP (DataBase of Single Nucleotide Polymorphisms). Our near-term and intermediate-term goals are to annotate and filter the sequence variants uncovered in the tumor-normal exomes with the aim of identifying somatic variant calls in protein-encoding genes for detailed follow up studies that will extend into future reporting periods.